The objective of the present paper was to evaluate the X-ray three-dimensional micro-computed tomography (X-microCT) method applied in assessing the trabecular structure in ovariectomized rats. Three-month-old female Sprague-Dawley rats (n = 30) were ovariectomized (OVX) or sham-operated (SHAM). OVX rats were treated with vehicle, or 17beta-estradiol (E2, positive control) for 3 months. For the conventional histomorphometric analysis, undecalcified sections were prepared and stained with the Li Chunhong technique to obtain high-contrast two-dimensional images. Prior to the histologic sectioning the samples were measured by X-microCT, providing a 14 microm resolution. The morphometric parameters computed by both methods in two or three dimensions, respectively, were bone volume over total volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N) and trabecular separation (Tb. Sp). Results showed that there were significant differences in the trabecular structure among three groups. In the OVX control group, the platelike structure was mostly resolved into a rodlike structure, with lots of the connecting rods missing. Whereas in OVX+E2 groups, this loss of trabecular bone mass and connectivity was prevented, with the results being nearly the same as those in the SHAM group. It was shown that highly significant correlations between conventional histology and X-microCT for BV/TV, Tb. Th, Tb. N and Tb. Sp were 0.984, 0.960, 0.995, and 0.988 in tibia, and 0.938, 0.968, 0.877 and 0.951 in lumbar. The high correlations between conventional histomorphometric and micro-tomographic analysis are very promising for the use of micro-tomographic imaging. X-microCT is a nondestructive, fast, and very precise procedure that allows the measurement of cancellous tissue in unprocessed biopsies or small bones, as well as a fully automatic determination of three-dimensional morphometric indices.